The long term goal of this project is to determine the human requirement for biotin in normal individuals and in individuals in certain circumstances in which biotin status may be impaired. This goal will be addressed in five specific aims. In specific aim #1, we will initially develop the capability to precisely measure the following: 1) two urinary organic acids (methylcitric acid and 3-hydroxypropionic acid) which are produced in increased quantities when activity of the biotin-dependent enzyme propionyl-CoA carboxylase is decreased, 2) lymphocyte carboxylase activities, 3) lymphocyte biotin content, and 4) serum odd-chain fatty acid content which also reflect decreased activity of propionyl-CoA carboxylase. Next we will determine the usefulness of these quantities as indicators of deficiency in rats whose biotin deficiency progresses from marginal to severe and then in marginally biotin deficient adult volunteers. Based on these studies, the best indicators will be chosen and used along with an established indicator - urinary 3-hydroxyisovaleric acid (3-HIA), an organic acid produced in increased quantifies when methylcrotonyl-CoA carboxylase is decreased - in two clinical studies: 1) in a radomized, bouble-blind biotin intervention study (Specific Aim #2), we will test the hypothesis that biotin status is impaired in individuals receiving long-term anticonvulsant therapy whenever urinary 3-HIA is abnormally increased; accordingly, urinary excretion of 3-HIA and other indicators of biotin-related metabolic derangement will return to normal with biotin supplementation. 2) In a second randomized, double-blind biotin intervention study (Specific Aim #3), we will test the hypothesis that biotin status is impaired whenever urinary 3-HIA is abnormally increased in pregnancy; accordingly, urinary 3-HIA and other biotin-related indicators of metabolic derangement will return to normal with biotin supplementation. Specific Aim #4 will determine whether biotin of similar severity to that observed in human pregnancy can cause significantly increased rates of fetal malformation in the mouse. In our pilot mouse study, marginal biotin deficiency in mouse dams that caused an increase in 3-HIA excretion similar to that seen in human pregnancy produced 100% incidence of cleft palate in the fetal mouse. Specific Aim #5 will determine the location and characterize the enzymes responsible for catalyzing the b-oxidation of biotin to the inactive metabolite bisnorbiotin. This catabolic process is of particular interest because increased biotin breakdown is induced by exposure to anticonvulsants and during pregnancy and may contribute to biotin deficiency in these circumstances.